In recent years, development of techniques to analyze genes in a vast number of samples that are stored in hospitals and research institutes in the forms of tissues and cells fixed with a fixative, such as formalin-fixed paraffin-embedded (FFPE) tissues, has been increasingly demanded. Since, especially as FFPE tissues, a vast amount of data on diseases obtained in the past have been accumulated, establishment of a technique that enables extraction, and analysis of expression, of genes extracted from FFPE tissues allows retrospective studies using tissues stored for a long period, largely contributing to future therapy and prophylaxis of diseases.
However, since degradation and fragmentation of RNAs extracted from fixed tissues and fixed cells such as FFPE samples proceed under general fixation conditions and storage conditions, it has been thought that gene expression analysis is difficult with such RNA. Further, formaldehyde (formalin), which is most commonly employed as a fixative, sometimes causes RNA-RNA or RNA-protein cross-linking, or addition of formaldehyde to RNA or modification of RNA with formaldehyde. In cases where RNA is in such a state, enzymatic reactions and/or chemical reactions hardly proceed, resulting in difficulty in analyzing gene expression. Therefore, a technology has been demanded by which analysis of gene expression can be carried out with an RNA sample showing extensive degradation and/or fragmentation, or with an RNA sample wherein cross-linking, addition and/or modification occurred. Further, it is very useful to judge, before gene expression analysis of such an RNA sample, the quality of the RNA sample by confirmation of the degrees of degradation/fragmentation, cross-linking and addition/modification to confirm whether or not the analysis is possible, for performing an accurate gene expression analysis and miRNA expression analysis, and hence a technique that enables such judgment has been demanded.
Japanese Translated PCT Patent Application Laid-open No. 2006-520603, JP 2005-224172 A, Japanese Translated Patent Application Laid-open No. 2007-515964 and Japanese Translated PCT Patent Application Laid-open No. 2008-541699 disclose techniques related to methods wherein gene expression analysis is performed after amplification of degraded RNA.
JP '603 provides a method, composition and kit related to amplification of a target polynucleotide to produce a large number of its copies. The amplification factor of mRNA per each time of amplification reaction is normally 50 to 100 or up to 250; 500 to 1000; or 500 to not less than 2000; and as much amplified RNA as possible is obtained from total RNA in an amount of less than a nanogram. However, in cases where such a method wherein RNA is amplified as much as possible from a small amount of RNA is employed, the amplification factor varies among genes, that is, the amplification bias increases, so that it can be said that the expression level of each gene cannot be analyzed accurately.
JP '172 relates to a method for using fragmented RNA or the like such as that contained in a stored fixed paraffin-embedded tissue material in comprehensive gene expression profiling, and provides a method of preparation of a sample that enables comprehensive amplification even from a sample composed of very small or extremely fragmented RNA. However, that method comprises a step of polyadenylation of fragmented RNA and, as a result, variation of the reaction in this step may largely influence the gene expression profile. Further, JP '172 discloses a novel linear RNA amplification method. In that method, a double-stranded cDNA having an anchor sequence at the 5′-end and an RNA polymerase promoter sequence at the 3′-end is synthesized, and cRNA is synthesized from the cDNA dependently on the RNA polymerase promoter sequence, followed by priming the anchor sequence to synthesize cDNA again from this cRNA, thereby amplifying a small amount of RNA obtained from a small amount of cells/tissue obtained by laser capture microdissection or cell sorting, to suppress deletion of the region of cDNA and cRNA corresponding to the 5′-region of mRNA that occurs every time the cDNA synthesis-cRNA synthesis cycle is repeated, which has been problematic in known amplification methods.
The methods in both JP '603 and JP '172 are described as methods showing no amplification bias, and it is thought that the bias per each cycle is smaller than that in conventional methods. However, since they focus on amplification of as much RNA as possible from a small amount of RNA, the amplification factor is considerably large. Accordingly, the bias may accumulate, resulting in occurrence of a large amplification bias.
JP '964 relates to a method for using fragmented RNA or the like such as that contained in a stored fixed paraffin-embedded tissue material in comprehensive gene expression profiling, and provides a method of preparation of a sample that enables comprehensive amplification even from a sample composed of very small or extremely fragmented RNA. However, that method comprises a step of polyadenylation of fragmented RNA, and, as a result, variation of the reaction in this step may largely influence the gene expression profile.
JP '699 discloses a composition and a method for amplifying a target in a degraded nucleic acid sample including a method for measuring the quality of nucleic acid in the nucleic acid sample, and also discloses a method for preparing a gene expression profile using a degraded RNA sample. The amplification efficiencies of amplicons (amplification products) having different sizes derived from the same gene are used as indices for evaluation of the quality, in view of the fact that the amplification efficiency of an amplicon having a large size decreases as degradation of the sample proceeds. That method performs multiple size PCR about each of ten to twenty and several genes. In cases where degradation of RNA has proceeded, amplification becomes less likely to occur as the PCR probe size increases, so that it is considered that the degree of degradation can be confirmed to a certain degree. However, since PCR amplification of a total of several ten genes is required per one RNA sample, it is thought that that method is difficult to actually carry out.
JP 2008-35779 A discloses a method wherein degradation index nucleic acid probes designed based on the base sequences of RNAs that are contained in the long-chain fraction if those are not degraded are loaded on a nucleic acid array, and an RNA sample prepared by fractionation of short chains from total RNA is hybridized with the nucleic acid array, followed by measuring, based on the presence/absence of signals from the degradation index nucleic acid probes, the degree of degradation of the RNA sample; and a technique related to the nucleic acid array for measurement of the degree of degradation of RNA. However, since the nucleic acid array is specified to short chain RNAs such as microRNA (miRNA), it is thought that application of the array to gene expression analysis is difficult. Further, in cases where RNA is extracted from a fixed tissue or fixed cell(s) such as FFPE, the amount of RNA which can be obtained is often small unlike cases of extraction of RNA from a cell(s) or a frozen tissue, and, under such conditions, it is not realistic to carry out an experiment using a sample in an amount of as much as several to several ten micrograms just for confirmation of the quality of the RNA sample. Also in view of the cost, that method can never be said to be a preferred method.
In JP 2008-43332 A, there is a description on a method for measuring the fragmentation level of nucleic acid. It discloses a kit for judging whether or not analysis of an RNA sample is possible, which judgment is made by measuring the amounts of 2 kinds of ribosomal RNA (18S and 28S) to determine the 28S/18S ratio. It is said that, when RNA is degraded, 28S is first degraded, and 18S is subsequently degraded. In that kit, in cases where 28S/18S is not more than 0.1, RNA is judged to have a bad quality. However, in terms of RNA extracted from a tissue or cell(s) fixed with a fixative, the RNA is generally degraded during fixation, and more-over, since fixed tissues and cells are normally stored at room temperature, the RNA is further degraded with time. Therefore, the above-described 2 kinds of ribosomal RNA often cannot be detected in RNA extracted from a formalin-fixed paraffin-embedded (FFPE) tissue, and, in such cases, according to the above-described standard for judgment of the quality of an RNA sample, most samples are determined to be unanalyzable. Thus, it is very difficult to use the kit for carrying out a retrospective study using a fixed tissue or fixed cell(s) stored for a long period.
Japanese Translated PCT Patent Application Laid-open No. 2009-501531 is a method of profiling of RNA wherein miRNA in a stored tissue containing degraded mRNA, which miRNA is bound to RISC(RNA-induced silencing complex) and not degraded, is released by heat treatment or the like, followed by detection of the miRNA by PCR amplification. Since the release of miRNA from RISC requires treatment at a high temperature of 95° C., the possibility of occurrence of degradation in this process cannot be excluded, and it does not mention a method for confirming the quality of RNA.
Further, in cases where a capillary electrophoresis system (e.g., “Bioanalyzer” manufactured by Agilent Technologies) is employed, RIN (RNA Integrity Number), which is a measurement standard developed by Agilent Technologies, is calculated as an index of RNA degradation. RIN is calculated based on the entire electropherogram of the RNA sample subjected to electrophoresis, and varies within the range of 0 to 10 (A. Schroeder, O. Mueller, S. Stocker, R. Salowsky, M. Leiber, M. Gassmann, S. Lightfoot, W. Menzel, M. Granzow and T. Ragg, “The RIN: an RNA integrity number for assigning integrity values to RNA measurements,” BMC Molecular Biology 7:3 (2006)). Bioanalyzer manufactured by Agilent Technologies is an apparatus commonly used for evaluation of the quality of nucleic acid and, in cases where this apparatus is employed, RIN is an index commonly used for representing the quality of RNA. However, when RNAs extracted from fixed tissues or fixed cells were analyzed with Bioanalyzer, even RNAs with clearly different electropherograms and various degradation behaviors show almost the same RIN values between 2 and 3. Therefore, there has been a possibility that RIN did not necessarily reflect the actual state of RNA. Means to judge whether or not RNA extracted from various tissues or cells fixed with a fixative can be subjected to the analysis is actually limited.
When RNA extracted from a tissue or cell(s) fixed with a fixative was to be analyzed, no method has been available to judge, simply and with a high probability, whether or not the extracted RNA is suitable for the analysis, and it was therefore difficult to know the appropriateness of data obtained by the analysis.